ABSTRACT
PURPOSE: To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. DESIGN: Evaluation of diagnostic test. PARTICIPANTS: Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. METHODS: We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. MAIN OUTCOME MEASURES: Collated clinical details and oculome molecular genetic results. RESULTS: The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia-anophthalmia-coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). CONCLUSIONS: The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.
Subject(s)
DNA Copy Number Variations/genetics , Eye Abnormalities/diagnosis , Eye Abnormalities/genetics , High-Throughput Nucleotide Sequencing/methods , Molecular Diagnostic Techniques , Mutation/genetics , Proteome/genetics , Adolescent , Child , Child, Preschool , Female , Genome, Human , Humans , Infant , Infant, Newborn , Male , PedigreeABSTRACT
BACKGROUND: Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development. METHODS AND FINDINGS: Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (nâ=â64) and control samples (nâ=â23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curveâ=â0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression. CONCLUSIONS: HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies. Please see later in the article for the Editors' Summary.
